Compositions of Carboxypolysaccharides and Polyalkylene Oxides Cross-Linked Using Polyhydroxyl Organic Anions

ABSTRACT

Ionically cross-linked compositions containing carboxypolysaccharides, polyalkylene oxides, polyhydroxyl organic anions, optionally divalent cations, and aqueous media are provided. Methods for manufacturing ionically cross-linked compositions of carboxypolysaccharides, polyalkylene oxides, polyhydroxyl organic anions, optionally divalent cations, and aqueous media are also provided. Such compositions can be used by placing then in proximity to a tissue in need of lubrication or for prevention of adhesions and adhesion reformation.

CLAIM OF PRIORITY

This Continuation application claims priority to U.S. patent applicationSer. No. 15/499,112 filed 27 Apr. 2017, (now U.S. Pat. No. 9,821,088issued 21 Nov. 2017), which is a Continuation of U.S. patent applicationSer. No. 15/215,950 filed 21 Jul. 2016 (now U.S. Pat. No. 9,636,434,issued 2 May 2017), which claims priority to U.S. ProvisionalApplication No. 62/350,907 filed 16 Jun. 2016 entitled “Compositions ofCarboxymethylcellulose and Polyethylene Oxide Cross-Linked UsingPolyhydroxyl Organic Anions, Samuel J. Falcone, Inventor. The abovepatents and applications are incorporated herein fully by reference.

FIELD OF THE DISCLOSURE

This disclosure relates generally to the manufacture and use ofcompositions comprising polyacids, polyalkylene oxides, and polyhydroxylorganic anions to form intermacromolecular complexes. Particularly, thisdisclosure relates to cross-linked compositions comprisingcarboxypolysaccharides, polyethers, and polyhydroxyl organic anions andthe use of those compositions.

BACKGROUND

Carboxymethylcellulose (CMC) is a water soluble, biocompatible andbioresorbable semi-synthesized polysaccharide. The safety ofcommercially available CMC having high purity has been identified andapproved by the Food and Drug Administration (FDA) for incorporationinto many products. CMC is able to react with various polymers by way ofelectrostatic interaction, ionic cross-linking, hydrogen bonding, Vander Waals interactions, and physical interpenetration. Because of itssafety, convenience and diversity of physico-chemical properties, CMChas demonstrated applications in the pharmaceutical, food and cosmeticindustries.

CMC is one type of carboxypolysaccharide (CPS). CPSs have also been usedin the manufacture of implantable polymers. CPSs are polymers made ofsaccharide monomers in which some of the hydroxyl (—OH) groups arereplaced with carboxyl groups (—COOH or COO—). Thus, CPSs such as CMChave some hydroxyl groups and some carboxyl groups present.Carboxylation can permit ionic interaction within a polymer chain or canpermit interaction between polymer chains, thereby forming a gel. Suchgels have been used for a variety of applications, including implantablemedical polymers.

Polyethers (PE) are polymers made of chains of ether residues(—CH₂—CH₂—). Polyethylene Oxide (PEO) is a polymer made with PE havinghydroxyl groups. PEOs can therefore form hydrogen bonds with carboxylgroups on CPSs, thereby forming a hydrogen-bonded composition. Moregenerally, polyalkylene oxides (POs or PAOs) can react in similarfashions with CPSs to form hydrogen bonded cross-linked compositions.

Adhesions are unwanted tissue growths occurring between layers ofadjacent bodily tissue or between tissues and internal organs. Adhesionscommonly form during the healing which follows surgical procedures, andwhen present, adhesions can prevent the normal motions of those tissuesand organs with respect to their neighboring structures.

The medical and scientific communities have studied ways of reducing theformation of post-surgical adhesions by the use of high molecular weightcarboxyl-containing biopolymers. These biopolymers can act as physicalbarriers to separate tissues from each other during healing, so thatadhesions between normally adjacent structures do not form.

SUMMARY

We have identified a new problem in the field, namely the need anddesire to provide ionically cross-linked biocompatible compositionshaving improved viscoelastic properties without increasing the body'sburden for eliminating the high molecular weight components of suchcompositions. Thus, there are several objects of the instant disclosure.

One object is to provide compositions and methods for manufacture anduse of CPS/PO/compositions cross-linked with polyhydroxyl organicanions.

One object is to provide compositions and methods which can be used toreduce the incidence of adhesion formation during and after surgery.This includes the prevention of de novo adhesion formation in primary orsecondary surgery.

One object is to prevent reformation of adhesions after a secondaryprocedure intended to eliminate the de novo adhesions which had formedafter a primary procedure.

One object is to provide antiadhesion compositions which remain at thesurgical site during the initial stages of critical wound healing.

One object of the disclosure is to provide antiadhesion compositionswhich can hydrate quickly in a controlled fashion.

One object of the disclosure is to provide compositions with desiredproperties with incorporated drugs, so that the drug can be deliveredlocally over a period of time to the surgical site.

One object of the disclosure is to provide compositions having improvedviscoelastic, antiadhesion, coatability, tissue adherence,anti-thrombogenicity or bioresorbability.

To achieve these and other objectives, in embodiments of the instantdisclosure one can carefully control the properties of antiadhesioncompositions by closely regulating the pH, amounts of carboxyl residues,polyether, cations, and polyhydroxyl anions within thecarboxypolysaccharide/polyether association complex, to closely controlthe degree of association between the polymers using polyhydroxylorganic anions.

In embodiments, polyhydroxyl organic anions such as gluconate can beused to provide intermolecular attraction, thereby providingcompositions having increased viscosity, and provide compositionviscosity control, compared to those made with CPS and PEO alone.

Creation of complexes in the form of compositions with desiredproperties is accomplished by varying the degree of hydrogen bondingbetween the polymers. This variation in properties is accomplished byvarying the amount of polyhydroxyl organic anion in the composition, thepH of the composition, the molecular weights of the polymers, thepercentage composition of the polymer mixture, and/or the degree ofsubstitution (d.s.) by carboxyl residues within the CMC or CPS, and thepresence and concentration(s) of polyhydroxyl anions.

To address the problems of the prior art antiadhesion compositions, wehave discovered new compositions based on association complexationbetween ionically associated carboxymethylcellulose (CMC) andhydrophilic polyethylene oxides (PEO). Cross-linked compositions of thisdisclosure can be made by dissolving CMC and PEO as dry powders in anaqueous solution, containing polyhydroxyl anions, and optionallymultivalent cations to provide cross-linking between the CMC and the PEOwith the polyhydroxyl organic anions acting as bridges for hydrogenbonding or van Der Waals interactions.

In embodiments of the disclosure, poly hydroxyl organic anions such asgluconate, and optionally, multivalent cations including Ca²⁺ can beused to provide intermolecular attraction, thereby providingcompositions having increased viscosity and provide viscosity control ofthe composition.

We unexpectedly found that CPS/PO gels made using poly hydroxyl organicanions exhibited viscoelastic properties very different from CPS/PO gelsmade with calcium chloride. In particular, we found that gels made usingcalcium chloride exhibited decreased viscosity with increasingconcentration of calcium chloride. In striking contrast, similar CPS/POgels made with polyhydroxyl organic anions exhibited increased viscosityat the same calcium ion concentration compared to gels made with calciumchloride. We further found, quite unexpectedly, that with increasingconcentration of polyhydroxyl organic anions, the viscosity of CPS/POgels increased with increasing calcium concentration. The increasedviscosity and increasing viscosity with increasing calcium concentrationof CPS/PO gels made with calcium/polyhydroxyl organic anions wascompletely unexpected based on the prior art.

The compositions of this disclosure can be used to inhibit post-surgicaladhesions, to decrease the consequences of arthritis or other disordersof joints, and/or to provide a lubricant for numerous medical and/orveterinary uses.

Additionally, in accordance with some aspects of the disclosure, drugscan be included in the compositions to deliver pharmacological compoundsdirectly to the tissues.

In certain embodiments, the compositions can be sterilized using thermalmethods, gamma irradiation, and ion beams which can alter the physicaland other properties of the components. Alternatively, in otherembodiments of this invention, the materials can be filter sterilized.

Manufacture of such compositions in the forms of membranes, beads,particulates, coatings and gels and some of their uses has beendescribed in U.S. Pat. Nos. 5,096,997, 6,017,301, 6,034,140, 6,133,325,6,566,345, 6,869,938 and 7,192,984, each patent expressly incorporatedherein fully by reference as if individually so incorporated.

Compositions of this invention can be useful for delivering drugs totissues. The sites of delivery of drugs using the compositions of thisinvention include, without limitation, skin, wounds, mucosa, internalorgans, endothelium, mesothelium, epithelium. In certain embodiments,buccal, optical, nasal, intestinal, anal, vaginal applications usingcompositions of this invention can be used.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be described with respect to the particularembodiments thereof. Other objects, features, and advantages of theinvention will become apparent with reference to the specification anddrawings in which:

FIG. 1 depicts a chemical diagram showing CMC and PEO polymerscross-linked with and gluconate. The portion of CMC shown includes twocellobiose units (each CMAG Unit having a FW of 242 Daltons).

FIG. 2 depicts graphs of viscosity (in Pascal·sec. “Pa·s”; verticalaxis) of CMC/PEO gels made with either calcium chloride (lower graph;filled squares) or calcium gluconate (upper graph; filled circles) atdifferent calcium concentrations (horizontal axis) as described inExamples 1 and 2.

FIG. 3 depicts graphs of viscosity (in Pascal·sec. “Pa·s”; verticalaxis) of CMC/PEO gels made with either calcium chloride (lower graph;filled squares) or calcium gluconate (upper graph; filled circles) atdifferent calcium concentrations (horizontal axis) as described inExamples 3 and 4.

FIG. 4 depicts graphs of viscosity (in Pascal·sec. “Pa·s”; verticalaxis) of CMC/PEO gels made with sodium gluconate (upper graph; filledcircles) at different calcium concentrations (horizontal axis) asdescribed in Examples 5 and 6.

DETAILED DESCRIPTION Definitions

The following definitions apply in general to the descriptions thatfollow. In certain cases, however, a term may be defined differently. Inthose cases, the proper definition will be provided.

The term “cross-linking” or “crosslinking” means covalent bonding of twopolymer chains together using a chemical reagent.

The term “carboxypolysaccharide” (CPS) means a polymer composed ofrepeating units of one or more monosaccharides, and wherein at least oneof the monosaccharide units has a hydroxyl residue substituted with acarboxyl residue.

The term “CMC” means sodium carboxymethylcellulose. “CMC A” is HerculesCMC grade 7H PH. and “CMC B” is Hercules grade 9M31F PH. PEO A is ofaverage molecular weight of 8000 Daltons and PEO B is of averagemolecular weight 4.4M Daltons.

The term “comprising” and “comprises” means includes enumeratedcomponents but is not limited to those components, and may includeadditional components.

The term “consisting of” means includes the enumerated components and islimited to those components.

The term “consisting essentially of” means includes the enumeratedcomponents and their equivalents.

The term “polyalkylene oxide” (“PO” or “PAO”) means non-ionic polymerscomprising alkylene oxide monomers. Examples of polyalkylene oxidesinclude polyethylene oxide (PEO), polypropylene oxide (PPO) andpolyethylene glycol (PEG), or block copolymers comprising PO and/or PPO.

The term “Polyethylene Oxide” (“PEO”) means the non-ionic polyetherpolymer composed of ethylene oxide monomers. The molecular weight of PEOas used herein is between 5,000 Daltons (“D”) and 8,000 kiloDaltons(“kD”).

The term “polyethylene glycol” (“PEG”) means a non-ionic polyetherpolymer being composed of ethylene oxide monomers, and having amolecular weight in the range of about 200 Daltons (“D”) to about 5000Daltons.

The term “Dalton” or “D” means a unit of molecular mass, where one D isequivalent to the mass of a proton.

The term “PEG” or “polyethylene glycol” means a polymer made ofrepeating units of compounds containing —(O—CH₂—CH₂)— but havingmolecular weights in the range of about 200 Daltons to about 5000 kDa.

The term “hydrogel” means a polymer matrix that swells in water but doesnot dissolve in water.

The term “transmural pressure” means the hydrostatic pressure inside thesac minus the hydrostatic pressure outside the sac.

The term “Laplace's laws” refer to two relationships between transmuralpressure, radius of a containment device (e.g., a “bag” or a “sac”) andthe wall stress. For a sphere, the wall tension ‘T’=Pressure “P” timesRadius “R,” or T=PR. For a cylinder, T=PR/2.

The term “viscosity” refers to a liquid-like property of a materialhaving a relatively high resistance to flow in response to an appliedforce. Viscosity is a measure of the viscous, or liquid-like, nature ofthe material.

The term “Storage Viscosity” (G′/ω) is the elastic modulus G′ divided bythe frequency (m).

The term “Dynamic Viscosity” (G″/ω) is the loss modulus (G″) divided bythe frequency (m).

The term “viscoelastic” means a property of polymeric materials thathave both elastic (solid-like) and viscous (liquid-like) properties.

The term “elasticity” means a rheological property defined as thecontribution of the elastic modulus, G′, to the overall stiffness of thematerial. Elasticity includes Percent Elasticity as a specific example.

The term “Percent Elasticity” is defined as to be equal to:100*G′/(G′+G″).

The term “pseudoplastic” means a rheological property of some polymersolutions characterized by a decrease in solution viscosity atincreasing shear rates.

The term “thixotropy” means a rheological property of some polymersolutions characterized by a time-dependent decrease in solutionviscosity at a constant shear.

The term “Fourier Transform Infrared Spectroscopy,” “FTIR Spectroscopy”or “FTIR” means an analytical technique that is used to detect variousorganic functional groups such as esters, ethers, etc. FTIR is based onabsorbance of infrared electromagnetic radiation by molecules (such asfunctional groups).

The term “carboxymethyl anhydroglucose unit” or “CMAG unit” or “CMAG” isan individual repeat unit of a polysaccharide polymer chain.

The term “phosphate buffered saline” or “PBS” means a solution of watercontaining a phosphate buffer.

The term “ionic cross-linking” or “ionic crosslinking” is a method ofcombining constituents through ionic bonds.

The term “physiological compatible aqueous solution” means a water-basedsolution containing other components rendering the solution able to beintroduced into a body without causing deleterious effects.Physiologically compatible aqueous solutions may have similar osmolarity(osmolality) as a body, may have a pH in the range of about 5.0 to about8.0.

The terms “% by weight” of a CPS or CPS/PO composition means a certainweight of the dry component dissolved in a certain weight of solvent.For example, a 10% solution by weight includes 10 grams (gms) of a CPSdissolved in 100 gms of solvent.

The term “% weight per volume” means a certain weight of dry componentdissolved in a solution having a total of a certain final volume. Forexample, a 10% weight/volume solution is one having 10 grams of soluteas part of 100 ml of solution.

Advantages of the Disclosure

Embodiments of the instant disclosure have significant advantages overprior art compositions. For example, Balazs et al., U.S. Pat. No.4,141,973 discloses the use of a hyaluronic acid (HA) fraction for theprevention of adhesions. However, because HA is relatively soluble andreadily degraded in vivo, it has a relatively short half-life in vivo of1 to 3 days, which limits its efficacy as an adhesion preventative.

Methyl cellulose and methyl cellulose derivatives are also known toreduce the formation of adhesions and scarring that may developfollowing surgery. (Thomas E. Elkins, et al., Adhesion Prevention bySolutions of Sodium Carboxymethylcellulose in the Rat, Part I, Fertilityand Sterility, Vol. 41, No. 6, June 1984; Thomas E. Elkins, M. D. etal., Adhesion Prevention by Solutions of Sodium Carboxymethylcellulosein the Rat, Part II, Fertility and Sterility, Vol. 41. No. 6, June 1984.However, these solutions are rapidly reabsorbed by the body anddisappear from the surgical site.

Additionally, solutions of polyethers can also decrease the incidence ofpost-surgical adhesions. Pennell et al., U.S. Pat. No. 4,993,585describes the use of polyethylene oxide in solutions of up to 15% todecrease formation of post-surgical adhesions. Pennell et al., U.S. Pat.No. 5,156,839 describes the use of mixtures of carboxymethylcellulose upto about 2.5% by weight, and polyethylene oxide, in concentrations of upto about 0.5% by weight in physiologically acceptable, pH neutralmixtures. Because of the neutral pH, these materials do not formassociation complexes, and thus, being soluble, are cleared from thebody within a short period of time.

The above-described solutions can have disadvantages in that they canhave short biological residence times and therefore may not remain atthe site of repair for sufficiently long times to have the desiredanti-adhesion effects. Therefore, antiadhesion membranes using certainpolymers have been made.

Although certain carboxypolysaccharide-containing membranes have beendescribed, prior membranes can have disadvantages for use to preventadhesions under certain conditions. Butler, U.S. Pat. No. 3,064,313describes the manufacture of films made of 100% carboxymethylcellulose(CMC) with a degree of substitution of 0.5 and below, made insoluble byacidifying the solution to pH of between 3 and 5, and then drying themixture at 70 EC to create a film. These films were not designed to beused as anti-adhesion barriers.

Anderson, U.S. Pat. No. 3,328,259 describes making films of 100%carboxymethylcellulose and polyethylene oxide, alkali metal salts, and aplasticizing agent for use as external bandages. These materials arerapidly soluble in plasma and water and thus would have a very shortresidence time as an intact film. Therefore, these compositions are notsuitable for alleviating surgical adhesions.

Smith et al., U.S. Pat. No. 3,387,061 describes insoluble associationcomplexes of carboxymethylcellulose and polyethylene oxide made bylowering the pH to below 3.5 and preferably below 3.0, and then dryingand baking the resulting precipitate (see Example XXXVIII). Thesemembranes were not designed for surgical use to alleviate adhesions.Such membranes are too insoluble, too stiff, and swell to little to beideal for preventing post-surgical adhesions.

Burns et al., U.S. Pat. No. 5,017,229 describes water insoluble filmsmade of hyaluronic acid, carboxymethyl cellulose, and a chemicalcross-linking agent. Because of the covalent cross-linking with acarbodiimide, these films need extensive cleaning procedures to get ridof the excess cross-linking agent; and because they are made without aplasticizer, they are too stiff and brittle to be ideally suited forpreventing adhesions—they do not readily conform to the shapes oftissues and organs of the body.

Thus, there is a need for antiadhesion compositions that can be usedunder a variety of different circumstances. D. Wiseman reviews the stateof the art of the field in Polymers for the Prevention of SurgicalAdhesions, In: Polymeric Site-specific Pharmacotherapy, A. J. Domb, Ed.,Wiley & Sons, (1994). A currently available antiadhesion gel is made ofionically cross-linked hyaluronic acid. (Huang et al., U.S. Pat. No.5,532,221).

Ionic cross-linking of polysaccharides is well documented in thechemical and patent literature (Morris and Norton, PolysaccharideAggregation in Solutions and Gels, Ch. 19, in Aggregation Processes inSolution, Wyn-Jones, E. and Gormally, J, Eds., Elsevier Sci. Publ. Co.NY (1983)). Each type of metal ion can be used to form gels of differentpolymers under specific conditions of pH, ionic strength, ionconcentration and concentrations of polymeric components. For example,alginate (a linear 1,4-linked beta-D-mannuronic acid, alpha-L-glucuronicacid polysaccharide) can form association structures betweenpolyglucuronate sequences in which divalent calcium ions can bind,leading to ordered structures and gel formation. Similar calcium bindingability is also demonstrated by pectin which has a poly-D-gidacturonatesequence. The order of selectivity of cations for pectins isBa²⁺>Sr²⁺>Ca²⁺. CMC also can bind to monovalent and divalent cations,and CMC solutions can gel with the addition of certain trivalent cations(Cellulose Gum, Hercules, Inc., page 23 (1984)).

Sayce et al. (U.S. Pat. No. 3,969,290) discloses an air freshener gelcomprising CMC and trivalent cations such as chromium or aluminum.

Smith (U.S. Pat. No. 3,757,786) describes synthetic surgical suturesmade from water-insoluble metal salts of cellulose ethers.

Shimizu et al. (U.S. Pat. No. 4,024,073) describe hydrogels consistingof water-soluble polymers such as dextran and starch chelated withcystine or lysine through polyvalent cations.

Mason et al. (U.S. Pat. No. 4,121,719) disclose CMC- and gumarabic-aluminum hydrogels used as phosphate binding agents in thetreatment of hyperphosphatemia.

U.S. Pat. No. 5,266,326 describes alginate gels made insoluble bycalcium chloride.

An antiadhesion gel is made of ionically cross-linked hyaluronic acid(Huang et al., U.S. Pat. No. 5,532,221). Cross-linking is created by theinclusion of polyvalent cations, such as ferric, aluminum or chromiumsalts. Hyaluronic acid (either from natural sources or bio-engineered)is quite expensive.

Pennell et al (U.S. Pat. No. 5,156,839) describes CMC solutionscontaining small amounts of high molecular weight PEO. In oneembodiment, Pennell describes covalently cross-linking gels usingdimethylolurea.

Schwartz et al. (U.S. Pat. Nos. 5,906,997, 6,017,301, 6,034,140, and6,133,325) describe compositions of carboxypolysaccharide polyethercomplexes and their use in reducing surgical adhesions.

Schwartz et al (U.S. Pat. No. 6,869,938) describes compositions ofpolyacids and plyalkylene oxides ionically cross-linked with calciumchloride for reducing adhesions.

Berg et al. (U.S. Pat. No. 7,192,984) describes compositions ofpolyacids and polyethers as dermal fillers.

Falcone et al. (U.S. Pat. Nos. 9,161,987, 9,345,809 and U.S. patentapplication Ser. No. 14/887,717) describe compositions of covalentlycrosslinked CMC/PEG and medical uses.

Therefore, the prior art discloses no compositions which are ideallysuited to the variety of uses of the instant disclosure.

DESCRIPTIONS OF SPECIFIC EMBODIMENTS

The descriptions of specific embodiments is intended to illustrateaspects of this disclosure, and is not intended to limit the scope ofthis disclosure. It can be appreciated that other applications of thecompositions described herein can be developed by persons of ordinaryskill in the art without undue experimentation. All of those embodimentsare considered part of this disclosure.

CPS/PO Compositions

This disclosure includes a variety of compositions having CPSs and POs,linked with polyhydroxyl organic anions and optionally multivalentcations. For example, CMCs are polymers composed of sugar residueslinked together, and one or more of which may have a carboxyl residueattached to the sugar moiety. There are three (3) potential sites forcarboxylation on each sugar residue of CMC. Because a carboxyl residuecan be chemically reactive, those locations on CMC are potential sitesfor derivatization. By controlling the degree of substitution (“DS” or“ds”) of the CMC or CPS, the number of active groups on the derivatizedCMC can be controlled.

FIG. 1 depicts a chemical diagram showing CMC and PEO polymerscross-linked with and gluconate. The portion of CMC shown includes twocellobiose units (each CMAG Unit having a FW of 242 Daltons).

FIG. 2 depicts graphs of viscosity (in Pascal·sec. “Pa·s”; verticalaxis) of CMC/PEO gals made with either calcium chloride (lower graph;filled squares) or calcium gluconate (upper graph; filled circles) atdifferent calcium concentrations (horizontal axis) as described inExamples 1 and 2.

FIG. 3 depicts graphs of viscosity (in Pascal·sec. “Pa·s”; verticalaxis) of CMC/PEO gels made with either calcium chloride (lower graph;filled squares) or calcium gluconate (upper graph; filled circles) atdifferent calcium concentrations (horizontal axis) as described inExamples 3 and 4.

FIG. 4 depicts graphs of viscosity (in Pascal·sec. “Pa·s”; verticalaxis) of CMC/PEO gels made with sodium gluconate (upper graph; filledcircles) at different calcium concentrations (horizontal axis) asdescribed in Examples 5 and 6.

PREPARATION OF CPS/PO IONICALLY-CROSSLINKED COMPOSITIONS

The CPS/PO compositions of this disclosure can be prepared into one ormore of several forms, including gels, membranes, beads, sponges andcoatings.

The types of CPSs that can be included in this disclosure are notlimited to CMC. Rather, carboxyethyl cellulose (CEC), hydroxymethylcellulose (HMC), cellulose and other cellulose derivatives can be used.

Uses of CPS/PO Compositions

CPS/PO compositions of this disclosure can be used for one or more ofthe following:

(1) as space filling materials, including those suitable forimplantation as load-bearing compositions placed at locations wherecompressive loads may occur, such as in the spinal cord, foraugmentation or replacement of the nucleus pulposus;(2) as delivery vehicles for controlled release of bioactive substances,such as drugs, growth factors, active peptides, genes, cells, clottingfactors such as thrombin, and antibiotics hormones includingepinephrine, steroids, anti-inflammatory agents and the like, andvasoconstrictors such as norepinephine and the like;(3) as delivery vehicles for the localized release of bioactivesubstances, such as drugs, growth factors, active peptides, genes,cells, clotting factors such as thrombin, and antibiotics, hormonesincluding epinephrine, steroids, anti-inflammatory agents and the like,and vasoconstrictors such as norepinephine and the like;(4) as binders for protein coupling and fatty absorption in both tissueengineering and food industries;(5) lubrication of joints and medical instruments;(6) tissue coating and tissue protection from fibrosis, neurotoxins,inflammatory mediators, free radicals and other harmful materials;(7) anti-adhesion compositions; and(8) as dermal fillers;

Useful properties include, but are not limited to bioadhesion,bioresorbability, antiadhesion, viscosity, and physicalinterpenetration.

Space Filling Materials

CPS/PO compositions of this disclosure can be particularly useful tofill voids in tissues resulting from disease or injury. For example,removal of a tumor during a surgical procedure can result in a loss oftissue volume. In situations in which organ or tissue function dependson the shape of the organ or tissue, CPS/PO compositions of thisdisclosure can be used to fill the void. Similarly, in injuries, such asexcavating injuries in which tissue volume is lost, CPS/PO clanpositionsof this invention can be used to fill those voids.

In other embodiments, space filling materials can be used to providebulk in internal locations. For example, in situations where a void hasbeen created as a result of removal of internal tissue (e.g., removal ofa sebacious cyst, bullet wound, removal of a localized tumor), in anarea not subject to large movements (e.g., the torso), an implant can bemade having a desired shape and having a desired elasticity. Such spacefilling materials, made according to the principles of this invention,can be highly biocompatible, having long residence times in the body.Such materials can be made in any particularly desired shape. Thus, ifthe void is irigular, the surgeon can shape the implant to match thevoid. After making a surgical incision through the skin, the implant isinserted and the wound sutured. Alternatively, the surgeon can inject acomposition comprising a gel having particles of CPS/PO materialtherein. After introduction into the void, the material can conform tothe shape of the void, thereby providing a uniform appearing structure.

In still further embodiments, space-filling materials can be containedwithin a biocompatible sac. For example, one can insert a CPS/POionically cross-linked composition into the spine to provide support insituations where the nucleus of a vertebral disk has become damaged. Byencasing CPS/PO compositions within a sac, the implanted material canresist compressive forces, and therefore can be used to avoid nervepinching, a common cause of pain in subjects with degenerating disks.

In still further embodiments, CPS/PO materials of this disclosure can bedried to form membranes. As described in U.S. Pat. No. 5,906,997(incorporated herein fully by reference), CPS/PO membranes can be madeby preparing a CPS/PO composition, and then drying the composition.

Load-Bearing Materials

Additionally, in situations in which tissue volume is lost throughdegeneration or other causes, CPS/PO compositions of this disclosure canbe used to decrease adverse effects of such tissue loss. In certainaspects, CPS/PO compositions of this disclosure can be used in loadhearing capacities. For example, voids in the bone (“bone voids”) of avertebral body (e.g., caused by surgical removal of a tumor ordegeneration of a vertebral disk nucleus) can result in pain, loss ofsensation and/or loss of motion or function, due in some cases, tocompression of spinal nerves. CPS/PO compositions can be made withvarying degrees of ionic cross-linking, and in cases in which there is ahigh degree of cross-linking, the compositions can support increasedloads. To make such compositions, the CPS can be used having a lowermolecular weight, so that higher concentrations of CPS may be usedresulting in more ether and carboxyl groups available for ioniccross-linking. Also, using POs having lower molecular weights, canincrease the number of cross-links (“cross-link density”) in the matrix.As the cross-link density increases, the ability of the matrix tosupport a load can increase. Thus, in certain aspects,highly-cross-linked CPS/PO compositions can be used as nucleusreplacements or to fill other bone voids.

In certain of these embodiments, a CPS/PO composition can be placed in abiocompatible sac or bag within the intervertebral space, other bonevoid where the disk and/or disk nucleus was present, or in otherlocations where containment of the composition of this invention isdesired. A bag can be made of a silicone-based polymer, such asSilastic™ or another, less deformable material, such as Mylar™ or othersuitable material. It can be desirable to use a bag that has sufficientstrength to resist breakage under the loads expected to be placed on thebag. Additionally, because both a CPS/PO composition and the walltension (under Laplace's law) can resist a load, these embodiments canbe used in situations in which relatively large loads are to be borne.

Thus, in some embodiments, a biologically compatible bag (which may havea “one-way” valve to permit introduction of material into the bag butwon't permit unwanted loss of material) of an appropriate size to fitwithin the intervertebral space or other location can be inserted in adeflated condition. Such insertion can minimize trauma to the vertebralbodies and the annulus. Once inserted into place, a CPS/PO ionicallycross-linked composition can be introduced into the bag using a needle.Once in the bag, the composition can act as a load-bearing structure,thereby replacing the lost or damaged nucleus. In embodiments in whichthere is frank loss of bone (e.g., due to removal of a tumor), a CPS/POionically cross-linked composition can be used to fill the void.Replacement CMC/PEO load bearing compositions generally can besurgically implanted. Alternatively, a biocompatible bag or sac can beinserted in a deflated condition as described above for nucleusreplacement, and then filled with a CPS/PO composition, which can act asa load-bearing material.

One can also introduce CPS/PO mixtures into a sac or bag, and thenintroduce multivalent cations and multivalent organic anions into thesac. Upon mixing, the CPS, PO, cations and anions can react with eachother, thereby forming the ionically cross-linked CPS/PO composition.

In some embodiments, multiple bags or sacs can be used. In particular,in situations in which high loads are to be expected (e.g., the knee,ankles or other lower extremities, or lower back), the use of multiple,small sacs can be employed to take advantage of the fact of thewell-known Laplace relationship between transmural pressure, radius andwall stress of a closed sac. As used herein the term “transmuralpressure” means the hydrostatic pressure inside the sac minus thehydrostatic pressure outside the sac. Thus, for a given transmuralpressure, a sac having a smaller radius will have lower wall stressplaced upon the sac. In contrast, a larger sac will have larger wallstress at the same transmural pressure because of the larger radius.

Based on the Laplace relationships (one for a sphere; T=PR and anotherfor a cylinder; T=PR/2), in certain embodiments of this disclosure, onecan use a series of small, cylindrical sacs, each inserted into a bonevoid and filled with CPS/PO composition of this disclosure. It can bedesirable to align the longitudinal axes of the small sacs in parallelwith the expected load. Thus, during polymerization, the small sacs cansupport the load better than a single sac having larger radius.

Additionally, in situations in which the annulus is damaged, one canintroduce one or more sacs in the intervertebral space, with one nearthe hole in the annulus. Thus, when filled, the sac near the hole in theannulus can effectively seal the hole and minimize further loss ofintervertebral material. In other embodiments, smaller sacs can beinserted and filled (to support the load) and a larger “plug” sac can beintroduced into the intervertebral space. If this plug sac is theninflated with a CPS/PO composition of this disclosure, the sac caneffectively plug the hole in the annulus and thus prevent the smallersacs from being extruded.

In additional aspects, CPS/PO compositions can be used to support loadswithin the skeletal system. For example, the spine is often a locationwhere degeneration, injury or disease can produce loss of structuralsupport. In particular, in conditions in which the disk is damaged,CPS/PO compositions of this invention can be readily used. In situationsin which the nucleus pulpous is partially or completely lost,compositions of this invention can be used to replace the lost tissue.In some of these embodiments, an elastic, relatively non-compressiblecomposition can be polymerized before insertion into the affected area.In other situations, one can administer a composition of this inventionprior to its polymerization, so that the composition polymerizes insitu. For example, in situations in which there is frank loss of bone,producing an irregularly shaped defect, a mixture of components of thisinvention can be injected. After polymerization, the composition can fitwell into the defect, thereby providing structural support.

In other situations, compositions of this invention can be placed withinone or more bags or sacs. These embodiments can have increasedload-bearing abilities, due to the facts that: (1) a composition can besupported against compression by the bag or sac, and/or (2) thecomposition has its own load-bearing abilities.

Vehicles for Controlled Delivery of Bioactive Substances

CPS/PO compositions of this disclosure can be used to deliver drugs,biologicals, nutrients or other biologically active agents (bioactivesubstances) to an animal. CPS/PO compositions can be made incorporatinga bioactive substance therein to provide a controlled-releasecomposition. In general, CPS/PO compositions having more cross-linkstend to retain bioactive substances more than compositions having fewercross-links. Such is the case for bioactive substances that are releasedfrom a CPS/PO composition by simple diffusion out of the matrix. In somecases in which the bioactive substance is large (e.g., protein), thebioactive substance may be released by a combination of simple diffusionout of the CPS/PO matrix and by release as the matrix is degraded in thebody.

Regardless of which type of release occurs, it can be appreciated thatthe release of bioactive agents can be controlled as desired by varyingthe composition of the CPS/PO composition of this disclosure. It is notintended that the type of bioactive substance be limited. Rather, anybioactive substance whose release is desired to be controlled can beeffectively delivered using CPS/PO compositions of this disclosure.

It can be readily appreciated that any number of drugs, biologicals andother chemical agents can be delivered using the CPS/PO composites ofthis disclosure.

It can also be appreciated that various hormones and steroids can bedelivered, as can other, systemically acting drugs, which can bedelivered transmucosally or transdermally. These include IgG, clottingfactors and enzymes for treating mucopolysaccharidosis or otherconditions.

Cardiovascular drugs include vasodilators such as β-adrenoreceptoragonists including terbutaline and low-dose epinephrine,α-adrenoreceptor antagonists including norepinephrine, high-doseepinephrine and the like, and vasodilators including nitroprusside andnitroglycerin.

Vehicles for Localized Release of Bioactive Substances

In situations in which local release of a bioactive substance isdesired, a CPS/PO composition of this disclosure can be useful. Suchsituations may apply during tissue healing after surgery, wherelocalized trauma to tissues produces localized inflammation. Thus, aCPS/PO composition of this disclosure may contain a vasoactive substanceto control bleeding (e.g., a vasoconstrictor, such as norepinephrine) orto promote hemostasis (e.g., a clotting factor). CPS/PO compositions ofthis disclosure can also be useful for localized delivery of toxicagents in chemotherapy. For example, after tumor resection surgery, itmay be desired to administer locally to the site, a CPS/PO compositionhaving a chemotherapeutic agent incorporated therein. Such localizedapplication may permit application of higher concentrations ofanti-tumor medications at the site needed, while reducing systemic sideeffects of traditional intravenous infection.

Certain agents can be advantageously used for local delivery, providingdesired concentration at a desired site, but while decreasingundesirable, systemic effects. Such agents include, but are not limitedto therapeutic proteins, such as thrombin to aid in attaining andmaintaining hemostasis, growth factors for bone, cartilage, skin andother tissue and cell types. Some of these peptide and protein growthfactors include bone morphogenic protein (BMP), epidermal growth factor(EGF), connective tissue growth factor (CTGF), platelet derived growthfactor (PDGF), angiotensin and related peptides, and RGD-containingpeptides.

Additionally, locally acting drugs include fungicides, histamine,antihistamine, anti-inflammatory drugs (corticosteroids, methotrexate),local anesthetics, angiogenesis promoting drugs (e.g., to treatcardiovascular disease, and anti-angiogenesis factors (e.g., to treattumors).

DNA-based therapeutics, including antisense DNA, gene therapeutics andRNA-based therapeutics are also suitably delivered using thecompositions of this invention. These agents can be used to eitherinhibit or promote transcription of endogenous genes, or alternatively,can provide exogenous gene products to promote local treatment.

Locally delivered chemotherapeutic agents can also be delivered. Theseinclude, by way of example only, antibiotics to treat microbialconditions, antifungal agents, antiparasitic agents, anti-neoplasticagents including alkylating agents, anti-metabolites and the like.

Binders for Protein Coupling

Other uses include more general uses of proteins, fats and otherbiological substances, whether bioactive or not. Thus, proteins can beincorporated into space filling CPS/PO compositions of this disclosure.Such proteins may include collagen, gelatin, or other proteins known inthe art.

Lubricants

CPS/PO compositions of this disclosure can be effective lubricants formedical instruments. In situations in which a medical instrument isinserted into a body, there is a likelihood of at least some tissuetrauma resulting. Trauma, even slight trauma, can cause tissue damage,and can result in unwanted effects. Such effects include bleeding,inflammation, adhesion formation or scarring. By coating a medicalinstrument with a CPS/PO composition of this disclosure prior toinsertion into the body, such trauma can be decreased.

Lubrication can be used for both acute and chronic uses. Thus, for anacute procedure, such as urethral catherization, a CPS/PO composition ofthis disclosure can be used to decrease pain and other discomfort aswell as ease insertion. By decreasing urethral trauma, use of CPS/POcompositions of this disclosure can decrease post-catheterization sideeffects, including decreasing pain on urination, urethral adhesions, anddecreased likelihood of infection.

CPS/PO compositions of this disclosure can also be effective tissuelubricants. In situations in which tissues traumatize adjacent tissues(e.g., in joints), compositions of this invention can be used todecrease friction and thereby decrease localized trauma. Thus, byinjecting a CPS/PO composition into an affected joint, pain can bereduced. By reducing friction anal pain, the usual cascade ofinflammation can be inhibited. By inhibiting inflammation, secondaryadverse effects of inflammation can be decreased. Such adverse effectscan be mediated by macrophages, leukocytes, mast cells, eosinophils, andmononuclear cells among others, each of which can produce bioactivesubstances that can make the situation worse. For example, mast cellscontain potent proteases (e.g., mast cell tryptase and mast cellchymotryptase) that can degrade normal tissue proteins. Additionally, aseries of interleukins can be released from neutrophils, macrophages andother inflammatory cells. Interleukins can be potent chemoattractivemolecules and can recruit other inflammatory cells locally to the area,and can thereby continue adverse side effects of tissue trauma. Finally,tissue trauma can activate neuropeptide-containing nerves (e.g.,c-fibers) known to contain substance P, which is a potent stimulus ofpain pathways. In addition to causing pain, substance P is a potentchemoattractant and stimulator of mast cells.

In certain embodiments, CPS/PO compositions of this disclosure can beused to lubricate joints, such as those in the spine. In particular,CPS/PO compositions can be used to lubricate facet joints betweenadjacent lateral spinous processes. Similarly, compositions of thisintention are useful as lubricants for other joints, including the knee,shoulder, elbows, wrists, ankles and hips.

Certain aspects of this disclosure include use of CPS/PO ionicallycross-linked compositions to provide lubrication for other joints andsoft tissues. In situations in which injury to bone, ligaments, tendons,fascia or other soft tissues has occurred, healing may not produce asmooth-functioning tissue. For example, damage to facet joints in thespine can result in abnormal alignment of vertebrae, which can lead tofurther damage to the disk (annulus or nucleus pulposus). Thus, a CPS/POcomposition of this disclosure can be injected between lateral spinousprocesses of adjacent vertebrae (which normally can slide past oneanother during normal movement). After such injection, the lateralspinous processes can be separated from each other, and the inherentlubrication afforded by the CPS/PO composition can decrease furtherirritation.

In a similar fashion, damage to tendons, ligaments and fascias canproduce pain, swelling and decreased function. This, insertion of aCPS/PO composition of this invention can improve mobility and candecrease the likelihood for further damage to the tissue.

Tissue Coatings: Antiadhersion Compositions

CPS/PO compositions of this disclosure have wide applicability as tissuecoating agents. CPS/PO compositions can be used as anti-adhesionpreparations. Adhesions are unwanted attachments of a tissue with anadjacent tissue. Adhesions commonly occur after surgery in which tissuesare damaged as a result of the surgery. Thus, CPS/PO compositions ofthis disclosure can be effectively used to provide a physical barrierbetween tissues that would otherwise tend to adhere to each other.CPS/PO compositions may be membranes, gels or sponges. Manyanti-adhesion uses are described in U.S. Pat. Nos. 5,096,997, 6,017,301,6,034,140, 6,133,325, 6,566,345, 6,869,938 and 7,192,984, each patentexpressly incorporated herein fully by reference as if individually soincorporated.

In still further embodiments, compositions of this invention can be usedas antiadhesion materials. Methods for using CPS/PO compositions forantiadhesion purposes are described in U.S. Pat. Nos. 5,906,997,6,017,301, 6,034,140, 6,133,325, 6,193,731, 6,869,938, and 7,192,984.Each of the aforementioned patents are expressly incorporated byreference as if separately so incorporated. It can be readilyappreciated that gels, membranes and other forms of the CPS/POcompositions of this invention can be used in similar ways.

Additionally, CPS/PO compositions of this disclosure can be useful tominimize joint pain. In numerous conditions, including arthritis,traumatic injury, degeneration of cartilage, and ligament damage, ajoint can become painful. A CPS/PO composition of this disclosure can beintroduced into an affected joint to provide lubrication and to protectadjacent tissues from damage caused by movement. For example, in theknee, a CPS/PO composition can be introduced during an arthroscopicprocedure. In situations in which the joint must bear a load (e.g.,knee, hip, ankle, vertebra), a CPS/PO composition can be made withparticularly high elasticity.

CPS/PO compositions can also be used to protect tissues from damage. Forexample, such compositions can protect peripheral nerves, tendons,ligaments, other soft tissues, synovial membranes, joints, and canthereby relieve pain.

For example, tendon and ligament injuries heal more slowly than to othertissues, in part because the blood flow to tendons and ligaments isreduced compared to tissues such as muscles, mesenteries, and the like.Furthermore, a tendon stress-related injury often is accompanied by astress injury to an adjacent ligament. Therefore, healing of bothtissues is required for a return to normal function. However, suchrecovery is often slow, and re-injury is common. Further, even whenhealed, tendons and ligaments tend to heal with scar tissue, which isnot smooth. Thus, even after healing, a previously injured tendon orligament may abrade adjacent tissues and cause either re-injury or slowrecovery processes.

In another example, in the spinal cord, damaged spinous processes orvertebral bodies may abrade adjacent tissues. Additionally, loss of avertebral nucleus can lead to compression of vertebral bodies and canresult in impingement of spinal nerves, often leading to pain and/orparalysis.

A further example can involve damage to peripheral nerves, where softtissue injury, trauma or inflammatory reactions can lead to pain or lossof nerve function. Application of a CPS/PO composition of thisdisclosure can decrease inflammatory responses, and therefore candecrease secondary damage caused by inflammatory reactions mediated by,for example, macrophages, leukocytes, mast cells or other types ofinflammatory cells.

Dermal Fillers

Augmentation of the skin can be an important factor in recovering frominjury or for cosmetic purposes. For example, with normal aging, skinmay become loose or creases can form, such as nasal-labial (nasolabial)folds. In the face, creases or lines may adversely affect a person'sself esteem or even a career. Thus, there has been a need forcompositions and methods that can diminish the appearance of creases orlines.

Further, there are situations in which loss of tissue can leave anindentation in the skin. For example surgical removal of a dermal cyst,lipoatrophy or removal of a solid tumor can result in loss of tissuevolume. In other cases, injuries, such as gunshot wounds, knife wounds,or other excavating injures may leave an indentation in the skin.Regardless of the cause, it can be desirable to provide a dermal fillerthat can increase the volume of tissue to provide a smoother or moreeven appearance.

Several compositions are available for such purposes. Collagen is oftenused as an injectable material for soft tissue augmentation.Additionally, numerous other materials, including proteins, fats,hyaluronic acid (HA), polyalcohols, and other polymers have been used asinjectable dermal fillers. However, non-cross linked, hydrophilicpolymers such as collagen, gelatin and HA have not performed well andmust be covalently cross-linked to remain in place to be effective. Oneexample is ZYDERM®, which is uncrosslinked bovine collagen, was noteffective as a dermal filler unless it was first cross-linked withglutaraldehyde to convert it to ZYPLAST®. Similarly, HA has not beensufficiently effective as a space filling material when injected orimplanted in the body unless it is first cross-linked.

Compositions of CPS and modified CPS have unique properties that allowsuch compositions to be injected into the skin to fill spaces and toprovide support where support is desired. One example for needed supportis dermal augmentation in the face where dermal and subdermal volume islost due to aging. CPSs including CMC have a unique property of being anelastic gel with unique physical properties such as dynamic, plastic andzero shear viscosity, tissue adhesiveness, cohesiveness and flowcharacteristics. In addition, it can achieve these properties withoutthe requirement of covalent cross-linking. CPSs including CMC areparticularly unique because chemical modifications of CMC increases thenumber of physical properties that make it an ideal injectable polymerfor human treatment. For example, change in the degree of substitutionhas a dramatic effect on thixotropy and on viscosity of the gel. Itsbiocompatability and viscoelastic properties make it uniquely useful forinjection into human skin where it becomes a space filling,biocompatible polymer.

Other polymers tested for their ability to perform as space filling gelsare polysaccharides that have been used for soft tissue filing areinferior to CPSs. For example, HA must be cross-linked to cause it tofunction as an elastic gel. Cross-linking limits its ability to beinjected through narrow gauge needles, because the cross-linkingconverts HA into particles. For example, RESTYLANE® is a productconsisting of cross-linked HA in a compatible solution.

Proteins used for dermal augmentation, such as collagen, also must becross-linked to perform well as dermal fillers. For example, ZYPLAST® isa cross-linked bovine collagen dermal filler.

CPSs can be a carrier for additional material for additional materialfor the skin, including hydrogel polymers such as PEO and emulsions.CPSs can be used to deliver drugs to the skin, such as antioxidants,retinol, vitamins and growth factors. Covalent cross-linking of polymersconverts them into particles that diminish their ability to deliveradditional polymers, liposomes, emulsions or other particulates.

Numerous substances have been tested over the years for augmenting softtissue in the dermis in the face to improve cosmesis by fillingdepressions in the skin (Klein and Elson, The History of Substances forSoft Tissue Augmentation, Dermatological Surgery 26:1096-1105, 2000).This is an area that continues to be studied as there has been noclearly superior material or product (Hotta, Dermal Fillers: The NextGeneration, Plastic Surgical Nursing 24(1):14-19, 2004). These fillersare prepared from several polymers including bovine collagen, porcinecollagen, chicken or bacteria fermented HA, gelatin, all of which arecross-linked covalently to reduce their dissolution time orimmunological reactions. Fillers also include autologous human collagen(cross-linked collagen from the patient), human cadaver dermis(cross-linked human collagen). Additional fillers are those that areinsoluble in the dermis, including PMMA beads, dPTFE (expandedpolytetrofluoroethylene), poly lactic acid, recombinant elastin, andthermoplastics that form gels when injected into humans (Klein andElson, The History of Substances for Soft Tissue Augmentation,Dermatological Surgery 26:1096-1105, 2000). More recently, ceramicparticles (U.S. Pat. No. 5,922,025) and also PMMA microspheres (Lemperleet al, Migration Studies and Histology of Injectable Microspheres ofDifferent Sizes in Mice, Plast. Reconstr. Surg 113(5):1380-1390 (2004)have been used for soft tissue augmentation.

Dermal fillers are used to fill scars, depressions and wrinkles. Dermalfiller substances have various inflammatory responses in the dermisincluding phagocytosis to foreign body reactions depending on thematerial (Lemperle et al., Human Histology and Persistence of VariousInjectable Filler Substances for Soft Tissue Augmentation, AestheticPlast. Surg. 27(5):354-366; discussion 367 (2003). One goal of dermalfillers it to temporarily augment the dermis to correct the surfacecontour of the skin without producing an unacceptable inflammatoryreaction, hypersensitivity reaction or foreign body reaction that causespain, redness or excessive scar formation for a period of time.

One of the first materials to be used for dermal augmentation isZYPLAST® derived form bovine collagen. A newer material used for thisapplication is RESTYLANE® derived from bacteria-produced HA. Becausechallenges include both biocompatibility and persistence in the skin,new dermal fillers are compared to one of the existing products such asZYPLAST® or RESTYLANE® (Narins et al., A Randomized, Double-Blind,Multicenter Comparison of the Efficacy and Tolerability of RestylaneVersus Zyplast for the Correction of Nasolabial Folds, Dermatol. Surg.29:588-595 (2003).

More recently, CMC has been used with polyethylene oxide (PEO) andmultivalent ions to produce ionically linked materials (U.S. Pat. No.7,192,984, incorporated herein fully by reference).

Compositions of this disclosure are particularly well suited as dermalfillers. As noted above, one of the difficulties with prior art dermalfillers is mismatching of the elasticities of the tissue and the dermalfiller. In situations where the tissue is relatively elastic and thedermal filler is relatively inelastic, lumps can appear where the tissuecan stretch, but the dermal filler does not. Conversely, in situationsin which the elasticity of the dermal filler is higher than that of thetissue, incomplete filling of voids can occur.

Thus, by selecting viscoelastic properties of a dermal filler toapproximate or match the elasticity of the tissue, a better void-fillingmaterial can be produced and used, while minimizing adverse effects oftissue-filler mismatching.

Additionally, as an individual ages, the elasticity of the skin tends todecrease. Thus, in subjects with less elastic skin, one might desirablyuse a dermal filler with lower elasticity then one might use in ayounger individual with more elastic skin. Similarly, certain tissuestend to have different elasticities or different mobilities. Forexample, the skin around facial muscles (e.g., nasolabial folds) may besubject to different stresses than other tissues (e.g., the lips). Thus,one can select dermal fillers having different viscoelastic propertiesfor use in the same subject.

Use of such dermal fillers depends upon the specific need. For example,when used to fill small wrinkles, such as nasolabial folds, or “crow'sfeet” around the eyes, dermal fillers in the form of a uniform gel orsmall particles can be desired. An advantage of using a uniform gel isthat these materials can be injected using very small needles, and canproduce a very smooth filling, particularly well suited for smoothingsmall lines. For use in somewhat larger lines (e.g., nasolabial folds),it can be desirable to use compositions comprising a gel havingparticles of CPS/PO. Such particles can be made according to methodsknown in the art, and can be made to have desired dimensions. For use innasolabial folds, the particles should be sufficiently small to passeasily through a small needle (e.g., a 25 or 30 gauge needle). Theremainder of the composition can be a CPS/PO gel having relatively lowerviscosity. After injection, the particles can further hydrate in thetissue, thereby forming a more uniform composition.

General Methods

In the following section, manufacture of CPS/PO compositions arepresented. However, it should be appreciated that CMC need not be theonly CPS used. Rather, any CPS can be used in manufacture of CPS/POcompositions in similar fashions without departing from the scope ofthis invention.

Manufacture of CPS/PO Compositions

To manufacture CPS/PO (e.g., CMC/PEO) compositions of this disclosure,generally the CPS is dissolved in aqueous medium, such as water, saline,phosphate-buffered saline, physiologically compatible aqueous solventsolution or other suitable medium. For example, dissolving a CPS inaqueous media is generally accomplished by adding a pre-weighed amountof powdered, dry CPS into a vessel containing the medium with stirring,such as with a vortex mixer until the CPS is completely dissolved. Insome embodiments, a CPS can be present in a concentration of from about1% by weight to about 30% by weight. In other embodiments, CMC can bepresent in a concentration of about 3% weight per volume to about 15% byweight per volume. The molecular weights of CPS can be in the range offrom about 50,000 D to about 1,000,000 D, alternatively from about 90 kDto about 700 kD.

To produce a CPS/PO composition, generally, pre-weighed amounts of CPSand PO are prepared, dissolved in an aqueous medium, containing apolyvalent cation (having a certain concentration) and a polyhydroxylorganic anion (having a certain concentration), with mixing, until thecomponents are well mixed. The solution after mixing is generally steamsterilized to directly prepare a sterile composition for use. Thus, aCPS/PO ionically cross-linked composition can be prepared and drawn intoa syringe, the syringe can then be sterilized (e.g., using steam). Whenthe reaction has occurred, the resulting material can be instilled intoa desired location through a small-gauge (e.g., 29 or 30 gauge) needle.In other embodiments, solutions of CPS and PO can be sterilized beforemixing.

In some embodiments, the solution can be a physiologically compatiblesolution, with biocompatible pH, ionic strength, and colloid osmoticpressure.

In certain embodiments, NaCl can be used in a concentration in the rangeof about 0.001% by weight or weight/volume to about 10% by weight orweight/volume, and alternatively from about 0.01 to about 5.0%. Incertain embodiments, isotonic saline can be used (e.g., about 0.9%).

A solution of multivalent cation (e.g., CaCl₂*H₂O) can be used inconcentrations of from about 0.001% weight/volume to about 50%weight/volume and in alternatives, from about 0.01% to about 10%. Inother embodiments, CPS/PO compositions can be made in phosphate bufferedsaline or other physiologically compatible medium.

Additionally, pH can be varied to produce compositions that have somehydrogen bonded components. Useful pH ranges can be in the range ofabout 6.0 to about 8.0. Adjusting pH can be accomplished by immersing aCMC/PEG composition in a buffer solution at the appropriate pH.

In other embodiments, CPS/PO compositions can be used precipitated andreconstituted in aqueous buffer solution.

Rheological Methods

Once a CPS/PO ionically cross-linked composition is prepared, itsviscoelastic properties can be readily determined using equipment andmethods known in the art. Small deformation oscillation measurementswere carried out with a Thermo Haake RS300 Rheometer, Newington, N.H.,in the cone and plate geometry. All measurements can be performed with a35 mm/1° titanium cone sensor at 25° C. The elastic modulus, G′, andloss modulus, G″, were obtained over a frequency range of 0.628-198rad/sec. Tan 8 was calculated as G″/G′.

In some embodiments, the CPS can be sodium carboxymethylcellulose (CMC)obtained from Hercules and polyethylene oxide) was obtained fromSigma-Aldrich Corporation.

According to manufacturer, CMC A has an average Mn of ˜700,000 Da andCMC B had a average Mn of ˜200,000 Da. Rheological measurements wereperformed on gels prepared at 30 mg/ml solids concentration in BupHModified Dulbecco's Phosphate Buffered Saline solution (PBS) purchasedfrom Pierce Chemical (catalog No. 28374). The solutions can be preparedby stirring or CMC into the PBS at room temperature for at least twohours. The resultant solutions were clear and colorless with no solidsevident and thus were used without filtration.

CPS/PO compositions can be sterilized using any conventional method,such as steam sterilization, irradiation or filtration.

Determination of Compressive Strength of CPS/PO Compositions

To determine the compressive strength of CPS/PO compositions, a suitablyshaped piece of material (e.g, 1 inch×1 inch×0.25 inches) can beprepared and placed on a surface such at a table. Once polymerizationhas occurred, a load (e.g., a known weight) can be placed on thecomposition. The weights can be progressively increased until thecomposition fractures. Alternatively, a composition can be placed in avise, with a pressure gauge inserted, and the load increasedprogressively until the composition fractures.

Sterilization of CPS/PO Compositions

As noted above, CPS/PO compositions may be conveniently sterilized usingheat. In some embodiments, the composition may be heated in an autoclaveor other steam producing apparatus. In some cases, it can be desirableto prepare a CPS/PO composition and then place it into a deliverydevice, such as a syringe. CPS/PO compositions can be made using a“3-step” process, in which: (1) a CPS/PO mixture is obtained, (2)multivalent cations and polyhydroxyl organic anions are added to startthe cross-linking reaction and (3) where the cross-linked material isprecipitated and reconstituted. Alternatively, a CPS/PO composition canbe made using a “one-step” process, in which the CPS/PO solution is madewith cations and anions and then placed in a delivery device forsterilization. After sterilization in situ, the CPS/PO composition isready to use.

One-Step Delivery

It can be appreciated that certain embodiments (e.g., “one-step”)embodiments can provide easy to produce, pre-sterilized compositions ina suitable delivery device (e.g., syringe). Pre-made CPS/POcompositions, having desirable elasticity, can be injected directly intothe site using a small gauge (e.g. 25, 26, 27, 28, 29 or 30 gauge)needle.

In each of the above situations, CPS/PO compositions of this inventioncan be beneficial.

Examples

The following examples are presented to illustrate certain embodimentsof this disclosure, and are not intended to limit the scope to theembodiments so illustrated. Rather, workers of skill in the art canmodify or adapt the teachings of this invention to make and use othervariations without undue experimentation. All of those embodiments areconsidered to be part of this disclosure.

Example 1. Preparation of CMC/PEO Gels in a CaCl₂/NaCl Salt Mixture:General Description

To a 400 ml polypentene beaker, fitted with a mechanical stirrer, weadded distilled, deionized water (DIW) (200 mL), CaCl₂ (see Table 1 foramounts), and NaCl (see Table 1 for amounts). After the salts weredissolved the solution was mixed at 700 rpm while a solid mixture of CMCA (6.66 g) and PEO B (0.74 g) were slowly added to the vortexingsolution. After 5 minutes the stirrer was set to 145 rpm for 2 hoursresulting in a clear viscous gel. The gel was filtered through a 100mesh filter under pressure and loaded into 20 mL polypropylene syringes.The syringes were loaded into autoclavable sterilization pouches andsteam sterilized. After sterilization the gel was set at roomtemperature overnight before measuring the viscosity, osmolality, andpH. Table 1 below presents results of this experiment. “Visc” meansviscosity, and “OSMO” means osmolality. Results are shown in Table 1below.

TABLE 1 Properties of CMC/PEO Gels made with CaCl₂ and NaCl PostSterililzed Values Gel Number [CaCl₂]mM [NaCl]mM Visc OSMO pH Gel 1-3 3369 268 275 6.16 Gel 1-2 16 99 364 315 6.26 Gel 1-1 8 99 431 280 6.35

Example 2. Preparation of a CMC/PEO Gel in a Calcium Gluconate/NaCl SaltMixture: General Description

To a 400 ml polypentene beaker, fitted with a mechanical stirrer, weadded distilled, deionized water (DIW) (200 mL), calcium gluconate (seeTable 2 for amounts), and NaCl (see Table 2 for amounts). After thesalts were dissolved the solution was mixed at 700 rpm while a solidmixture of CMC A (6.66 g) and PEO B (0.74 g) were slowly added to thevortexing solution. After 5 minutes the stirrer was set to 145 rpm for 2hours resulting in a clear viscous gel. The gel was filtered through a100 mesh filter under pressure and loaded into 20 mL polypropylenesyringes. The syringes were loaded into autoclavable sterilizationpouches and steam sterilized. After sterilization the gel was set atroom temperature overnight before measuring the viscosity, osmolality,and pH. After sterilization the gel was set at room temperatureovernight before measuring the viscosity, osmolality, and pH. Table 2below presents results of this experiment. “Visc” means viscosity,“OSMO” means osmolality, and “CaClu” means calcium gluconate.

TABLE 2 Properties of CMC/PEO Gels made with CaGluconate and NaCl PostSterilized Values Gel Number [CaGlu] [NaCl] Visc OSMO pH 2-3 33 69 925296 6.37 2-2 16 99 884 314 6.41 1-2 1-1? 8 99 642 284 6.42

Viscosities of the post-sterilized compositions of Examples 1 and 2 aredepicted in FIG. 2.

As shown in Table 1 and FIG. 2, the viscosity of compositions of thisdisclosure are very different from those of the prior art. Results ofthe experiments in Example 1 show notable properties. First, withincreasing calcium concentration, the viscosity of CaCl₂-containingmaterials decreases. Such behavior is characteristic of compositions ofCPS and PO ionically cross-linked with CaCl₂. This property is shared bythe compositions disclosed by Schwartz et al. (U.S. Pat. No. 6,869,938.

In contrast to the observed decrease in viscosity with increasingcalcium ion concentration, compositions of the instant disclosureexhibited two properties shown in Example 2. First, as shown in FIG. 2,at the same calcium ion concentration, the viscosity of calciumgluconate-containing compositions is higher that for compositionsionically cross-linked using CaCl₂. Second, and most surprisingly, withincreasing calcium ion concentration, the viscosity of calciumgluconate-containing compositions increases, so that the viscosity ofthe gluconate-containing compositions is substantially higher than theviscosity of compositions cross-linked using CaCl₂ (or calcium ions, perse).

These effects represent a result that is completely unexpected based onthe prior art. These effects represent an advantageous and excellenteffect, and render compositions of the instant disclosure highlysuitable for the uses to which such compositions can be used. Forexample, using the disclosures herein, it is possible to produce acomposition having a desired viscosity without using increased amountsof CPS or PO. Because CPS and PO are eliminated according tophysiological processes, one may be able to use a high-viscositymaterial that does not overburden the physiological processes, and insome cases, may permit a shorter residence time in the body. Such afeature may be particularly useful in acute situations in which theresidence time of the composition need not be long.

Example 3. Preparation of a CMC/PEO Gels in a CaCl₂/NaCl Salt MixtureGeneral Description

To a 400 ml polypentene beaker, fitted with a mechanical stirrer, weadded distilled, deionized water (DIW) (200 mL), CaCl2 (see Table 3 foramounts), and NaCl (see Table 3 for amounts). After the salts weredissolved the solution was mixed at 700 rpm while a solid mixture of CMCA (7.21 g) and PEO A (0.19 g) were added to the vortexing solution.After 5 minutes the stirrer was set to 145 rpm for 2 hours resulting ina clear viscous gel. The gel was filtered through a 100μ mesh filterunder pressure and loaded into 20 mL polypropylene syringes. Thesyringes were loaded into autoclavable sterilization pouches and steamsterilized. After sterilization the gel was set at room temperatureovernight before measuring the viscosity, osmolality, and pH. Resultsare shown in Table 3 below.

TABLE 3 Properties of CMC/PEO Gels made with CaCl₂ and NaCl. PostSterilized Values Gel Number [CaCl₂]mM [NaCl]mM Visc OSMO pH Gel 3-3 3369 294 280 6.55 Gel 3-2 16 99 393 310 6.60 Gel 3-1 8 99 461 295 6.57

Example 4. Preparation of CMC/PEO Gels in a Calcium Gluconate/NaCl SaltMixture General Description

To a 400 ml polypentene beaker, fitted with a mechanical stirrer, weadded distilled, deionized water (DIW) (200 mL), calcium gluconate (seeTable 4 for amounts), and NaCl (see Table 4 for amounts). After thesalts were dissolved the solution was mixed at 700 rpm while a solidmixture of CMC A (7.21 g) and PEO A (0.19 g) were slowly added to thevortexing solution. After 5 minutes the stirrer was set to 145 rpm for 2hours resulting in a clear viscous gel. The gel was filtered through a100p mesh filter under pressure and loaded into 20 mL polypropylenesyringes. The syringes were loaded into autoclavable sterilizationpouches and steam sterilized. After sterilization the gel was set atroom temperature overnight before measuring the viscosity, osmolality,and pH. After sterilization the gel was set at room temperatureovernight before measuring the viscosity, osmolality, and pH. Resultsare shown in Table 4 below.

TABLE 4 Properties of CMC/PEO Gels made with CaGluconate and NaCl. PostSterilized Values Gel Number [CaGlu] mM [NaCl] mM Visc OSMO pH 4-3 33 691015 298 6.77 4-2 16 99 948 310 6.60 4-1 8 99 705 295 6.66

It can be readily appreciated that the presence of gluconatesubstantially increased the viscosity of gels containing thispolyhydroxyl anion.

Example 5. Preparation of CMC/PEO Gels in a Sodium Gluconate/NaCl SaltMixture: General Description

To a 400 ml polypentene beaker, fitted with a mechanical stirrer, weadded distilled, deionized water (DIW) (200 mL), sodium gluconate (seeTable 5 for amounts), and NaCl (see Table 5 for amounts). After thesalts were dissolved the solution was mixed at 700 rpm while a solidmixture of CMC A (6.66 g) and PEO B (0.74 g) were slowly added to thevortexing solution. After 5 minutes the stirrer was set to 145 rpm for 2hours resulting in a clear viscous gel. The gel was filtered through a100p mesh filter under pressure and loaded into 20 mL polypropylenesyringes. The syringes were loaded into autoclavable sterilizationpouches and steam sterilized. After sterilization the gel was set atroom temperature overnight before measuring the viscosity, osmolality,and pH. After sterilization the gel was set at room temperatureovernight before measuring the viscosity, osmolality, and pH. Resultsare shown in Table 5 below.

TABLE 5 Properties of CMC/PEO Gels made with NaGluconate and NaCl PostSterilized Values Gel Number [NaGlu] mM [NaCl] mM Visc OSMO pH 5-3 33 691003 300 6.55 5-2 16 99 861 312 6.52 5-1 8 99 700 296 6.57

It can be readily appreciated that the effect of gluconate to increaseviscosity of CPS/PO gels is not dependent on the cation present in themixture. Thus, sodium gluconate (Table 5) and calcium gluconate (Table4) show similar increases in viscosity.

Example 6. Preparation of CMC/PEO Gels in a Sodium Gluconate/NaCl SaltMixture: General Description

To a 400 ml polypentene beaker, fitted with a mechanical stirrer, weadded distilled, deionized water (DIW) (200 mL), sodium gluconate (seeTable 6 for amounts), and NaCl (see Table 6 for amounts). After thesalts were dissolved the solution was mixed at 700 rpm while a solidmixture of CMC A (7.21 g) and PEO A (0.19 g) were slowly added to thevortexing solution. After 5 minutes the stirrer was set to 145 rpm for 2hours resulting in a clear viscous gel. The gel was filtered through a100p mesh filter under pressure and loaded into 20 mL polypropylenesyringes. The syringes were loaded into autoclavable sterilizationpouches and steam sterilized. After sterilization the gel was set atroom temperature overnight before measuring the viscosity, osmolality,and pH. After sterilization the gel was set at room temperatureovernight before measuring the viscosity, osmolality, and pH. Resultsare shown in Table 6 below.

TABLE 6 Properties of CMC/PEO Gels in a Sodium Gluconate/NaCl SaltMixture Post Sterilized Values Gel Number [NaGlu] mM [NaCl] mM Visc OSMOpH 6-3 33 69 1010 315 6.65 6-2 16 99 870 300 6.61 6-1 8 99 725 299 6.657

1-58. (canceled)
 59. A composition, comprising: a carboxypolysaccharide(CPS), said CPS being hyaluronic acid (HA), a polyalkylene oxide (PO),said CPS and said PO ionically cross-linked using a polyhydroxyl organicanion (PHA), and a physiologically compatible aqueous solution.
 60. Thecomposition of claim 59, said PO being polyethylene oxide (PEO).
 61. Thecomposition of claim 59, said PHA being gluconate.
 62. The compositionof claim 59, further comprising a cation.
 63. The composition of claim62, said cation being calcium (Ca⁺²).
 64. The composition of claim 62,said cation being sodium (Na+).
 65. The composition of claim 59, said POhaving an average molecular weight of between about 5000 Daltons andabout k8000 Daltons.
 66. The composition of claim 59, said compositionhaving a ratio of HA, PO, and PHA to said physiologically compatibleaqueous medium being in the range of about 1% by weight solids to totalsolution to about 30% by weight solids to total solution.
 67. A method,comprising providing components: a hyaluronic acid (HA), a PO, a PHA,and an aqueous solution; and dissolving said HA, PO, and PHA in saidaqueous solution, thereby forming an ionically cross-linked composition.68. The method of claim 66, further comprising a cation.
 69. The methodof claim 67, said cation being calcium (Ca⁺²).
 70. The method of claim68, said cation being sodium (Na+).
 71. The method of claim 66, furthercomprising sterilizing said composition.
 72. A method, comprising:providing a composition of claim 59; and inserting said composition intoa portion of a body in need thereof.
 73. The method of claim 72, saidneed comprising one or more of: anti-adhesion, tissue lubrication,lubrication of an instrument, and a space-filling.
 74. The method ofclaim 72, said composition comprising HA, PEO A, gluconate, and anaqueous medium.
 75. The method of claim 74, said aqueous solution beinga physiologically compatible medium.